PROJECT SUMMARY The aim of this proposal is to develop a rapid, low-cost, and easy-to-use POC diagnostic test for Zika virus (ZIKV). ZIKV is an emerging mosquito-borne Flavivirus that has gained global attention due to new evidence that it is linked to birth defect microcephaly. Additionally, health experts are also concerned about the link between the virus and Guillain-Barr syndrome (GBS). President Obama announced in early February of 2016 that he is asking Congress for more than $1.8 billion in emergency funding to expand the United States? efforts to combat Zika. One of the most vital tools necessary to confront an outbreak like the Zika virus is a reliable, accurate, and easily accessible diagnostic test. The issue with existing tests is that they often fail to distinguish between Zika and similar viruses to which people living in Central and South America are frequently exposed. The CDC has recommended the use of molecular detection (e.g., reverse transcription polymerase chain reaction or RT-PCR) to identify Zika virus in suspected cases. While CDC and qualified state health department labs can perform the RT-PCR ZIKV test, these labs use highly sophisticated instruments and turnaround times are typically weeks. To this end, we will collaborate with internationally known arboviruses expert and leading US ZIKV researcher Professor Scott Weaver at the University of Texas Medical Branch (UTMB) to develop a low-cost POC system for testing ZIKVs infection in non-traditional healthcare settings in the US and Americas. This device uses a wind-up music box to drive an innovatively designed plastic gear set to automate nucleic acid extraction in pre-filled cartridges and to elute the purified RNA directly into real-time master mix of amplification assays such as reverse transcription recombinase polymerase amplification reaction (RT-RPA) or polymerase chain reactions (RT-PCR). The gear set and the disposable cartridge can be enclosed for biosafety and contamination prevention. This portable system lacks complicated microfluidics, pumps or other sensitive/high cost components, thus making it suitable for resource-limited countries. The simple sample-to-answer process requires minimal training and informs healthcare workers of patients? diagnostic statuses. In Phase I, we will first demonstrate quality RNA extraction from spiked serum and urine samples in 10 min using pre-filled cartridges and the gear set. We will also demonstrate that the eluted RNA samples can be rapidly amplified to confirm the presence of ZIKV RNA using isothermal real-time RT-RPA assay. We aim to complete real-time RT-RPA amplification reactions in less than 15 min. The fluorescent signal produced from amplification will be monitored using LEDs for excitation and a smartphone camera for emission monitoring and analysis. Near the end of Phase I, our collaborator will validate our device for ZIKV detection. The successful development and commercialization of our technology will lead to molecular diagnostics being implemented in non-laboratory settings and will assist resource-limited populations to get tested for Zika. In short, our portable system will be pertinent to the ongoing ZIKV global health emergency.